Conjugate vaccine targeting a disease-causing biological protein

ABSTRACT

The present invention provides a vaccine containing a complex of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and an epitope of a disease-causing biological protein such as DPP4, IL-17A, IgE, S100A9 or PCSK9, which vaccine uses a less antigenic carrier protein and is capable of inducing antibody production to serve as an effective vaccine.

TECHNICAL FIELD

The present invention relates to a conjugate vaccine targeting adisease-causing biological protein.

BACKGROUND ART

Vaccine techniques have been long used for the prevention of infectionssuch as smallpox, and have recently been attempted to be used for thetreatment of cancer, life-style related diseases, etc. Some therapeuticvaccines use a carrier protein conjugated to an epitope of a targetprotein, which stimulates immune responses, for example, the inductionof antibodies against the target protein. Known examples of such acarrier protein include KLH (keyhole limpet hemocyanin), OVA (ovalbumin)and BSA (bovine serum albumin). For example, for the treatment ofsystemic lupus erythematosus (SLE), the administration of a vaccinecomposed of a modified form of INF-α as an antigen moiety conjugated toKLH has been attempted (Non Patent Literature 1).

However, KLH, OVA, BSA and other carrier proteins are antigenic inthemselves, and this antigenicity can sometimes pose a problem in thecase where a moderately antigenic epitope conjugated to such a carrierprotein is used for immunization (Non Patent Literature 2).

The present inventors disclose the use of a conjugate of a DPP4 epitopepeptide and KLH for the prevention or treatment of diabetes mellitus(Patent Literature 1). In addition, the present inventors disclose theuse of a DNA vaccine containing a polynucleotide encoding an IL-17Aepitope peptide and a polynucleotide encoding a core antigen polypeptideof hepatitis B virus for the prevention or treatment of diseases inwhich IL-17A is involved as a precipitating factor (e.g., systemic lupuserythematosus (SLE), articular rheumatism, etc.) (Patent Literature 2).

CITATION LIST Patent Literature

-   Patent Literature 1: WO2015/033831-   Patent Literature 2: WO2015/099167-   Non Patent Literature 1: ARTHRITIS & RHEUMATISM Vol. 65, No. 2,    February 2013, pp 447-456.-   Non Patent Literature 2: N. E. van Houten, M. B. Zwick, A. Menendez,    and J. K. Scott Vaccine. 2006 May 8; 24(19): 4188-4200.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a vaccine whichcontains a complex of a less antigenic carrier protein conjugated to anepitope of a disease-causing biological protein and is capable ofinducing antibody production to serve as an effective vaccine.

Solution to Problem

The present invention includes the following to achieve theabove-mentioned object.

(1) A vaccine comprising a complex of a peptide consisting of an aminoacid sequence that is the same or substantially the same as the aminoacid sequence of SEQ ID NO: 1 and an epitope of a disease-causingbiological protein.(2) The vaccine according to the above (1), wherein the biologicalprotein is one kind selected from the group consisting of DPP4, IL-17A,IgE, S100A9 and PCSK9.(3) The vaccine according to the above (2), wherein the epitope ofIL-17A is a peptide consisting of the amino acid sequence of any of SEQID NOs: 10, 11 and 29 to 36, or a peptide consisting of an amino acidsequence that is the same as the amino acid sequence of any of SEQ IDNOs: 10, 11 and 29 to 36 except for 1 or 2 amino acid deletions,substitutions or additions.(4) The vaccine according to the above (2), wherein the epitope of DPP4is a peptide consisting of the amino acid sequence of any of SEQ ID NOs:2 to 9, or a peptide consisting of an amino acid sequence that is thesame as the amino acid sequence of any of SEQ ID NOs: 2 to 9 except for1 or 2 amino acid deletions, substitutions or additions.(5) The vaccine according to the above (2), wherein the epitope of IgEis a peptide consisting of the amino acid sequence of SEQ ID NO: 12, ora peptide consisting of an amino acid sequence that is the same as theamino acid sequence of SEQ ID NO: 12 except for 1 or 2 amino aciddeletions, substitutions or additions.(6) The vaccine according to the above (2), wherein the epitope ofS100A9 is a peptide consisting of the amino acid sequence of SEQ ID NO:13, or a peptide consisting of an amino acid sequence that is the sameas the amino acid sequence of SEQ ID NO: 13 except for 1 or 2 amino aciddeletions, substitutions or additions.(7) The vaccine according to the above (2), wherein the epitope of PCSK9is a peptide consisting of the amino acid sequence of any of SEQ ID NOs:14 to 16, or a peptide consisting of an amino acid sequence that is thesame as the amino acid sequence of any of SEQ ID NOs: 14 to 16 exceptfor 1 or 2 amino acid deletions, substitutions or additions.(8) The vaccine according to any one of the above (1) to (7), whereinthe epitope of the biological protein is conjugated to the peptideconsisting of the amino acid sequence of SEQ ID NO: 1 via ε-aminocaproicacid.(9) The vaccine according to any one of the above (1) to (8), whereinthe amino acid at the N-terminus of the complex is acetylated.(10) The vaccine according to any one of the above (1) to (9), whereinthe amino acid at the C-terminus of the complex is amidated.(11) An immunogenic composition comprising a complex of a peptideconsisting of the amino acid sequence of SEQ ID NO: 1 and an epitope ofa disease-causing biological protein.(12) The immunogenic composition according to the above (11), whereinthe biological protein is one kind selected from the group consisting ofDPP4, IL-17A, IgE, S100A9 and PCSK9.(13) A method for preventing or treating a disease caused by abiological protein, the method comprising administering, to an animal,an effective amount of a complex of a peptide consisting of an aminoacid sequence that is the same or substantially the same as the aminoacid sequence of SEQ ID NO: 1 and an epitope of the biological protein.(14) A complex for use in prevention or treatment of a disease caused bya biological protein, the complex of a peptide consisting of an aminoacid sequence that is the same or substantially the same as the aminoacid sequence of SEQ ID NO: 1 and an epitope of the biological protein.(15) Use of a complex of a peptide consisting of an amino acid sequencethat is the same or substantially the same as the amino acid sequence ofSEQ ID NO: 1 and an epitope of a disease-causing biological protein, forproduction of a medicament for prevention or treatment of a diseasecaused by the biological protein.

Advantageous Effects of Invention

The present invention provides a vaccine which contains a complex of aless antigenic carrier protein conjugated to an epitope of adisease-causing biological protein and is capable of inducing antibodyproduction to serve as an effective vaccine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the antibody production-inducing effect of an OSK-1-DPP4conjugate composed of an OSK-1 peptide (SEQ ID NO: 1) conjugated to amouse DPP4 epitope peptide (SEQ ID NO: 17) via an ε-Acp linker.

FIG. 2 shows the comparison of the antibody production-inducing effectsof the OSK-1-DPP4 conjugate and a conjugate of KLH and a mouse DPP4epitope peptide (SEQ ID NO: 17) (KLH-DPP4).

FIG. 3 shows the results of the analysis of the IgG subclass ofantibodies produced by vaccination with the OSK-1-DPP4 conjugate incomparison with a KLH-DPP4 conjugate plus Alum group and a KLH-DPP4conjugate plus OSK-1 group.

FIG. 4 shows the antitumor growth effect of an OSK-1-WT1 conjugatecomposed of the OSK-1 peptide (SEQ ID NO: 1) conjugated to a WT1 peptide(SEQ ID NO: 28) via an ε-Acp linker in mice having undergone vaccinationwith the OSK-1-WT1 conjugate followed by subcutaneous implantation ofcancer cells.

FIG. 5 shows the survival benefit of the OSK-1-WT1 conjugate in micehaving undergone vaccination with the OSK-1-WT1 conjugate followed bysubcutaneous implantation of cancer cells.

FIG. 6 shows the antitumor growth effect of the OSK-1-WT1 conjugate inmice having undergone subcutaneous implantation of cancer cells and thefirst vaccination with the OSK-1-WT1 conjugate on the same day.

FIG. 7 shows the survival benefit of the OSK-1-WT1 conjugate in micehaving undergone subcutaneous implantation of cancer cells and the firstvaccination with the OSK-1-WT1 conjugate on the same day.

FIG. 8 shows the comparison of OSK-1-DPP4 conjugate-induced IgEproduction against a target protein with KLH-DPP4 conjugate-induced IgEproduction against the target protein.

FIG. 9 shows (A) the results of amino acid analysis and (B) the resultsof HPLC analysis of an OSK-1-DPP4 conjugate composed of the OSK-1peptide conjugated to a mouse IL-17A epitope peptide (SEQ ID NO: 21) viaan ε-Acp linker.

FIG. 10 shows (A) the results of amino acid analysis and (B) the resultsof HPLC analysis of an OSK-1-DPP4 conjugate composed of the OSK-1peptide conjugated to a mouse IL-17A epitope peptide (SEQ ID NO: 31) viaan ε-Acp linker.

FIG. 11 shows (A) the results of amino acid analysis and (B) the resultsof HPLC analysis of an OSK-1-DPP4 conjugate composed of the OSK-1peptide conjugated to a mouse IL-17A epitope peptide (SEQ ID NO: 32) viaan ε-Acp linker.

FIG. 12 shows (A) the results of amino acid analysis and (B) the resultsof HPLC analysis of an OSK-1-DPP4 conjugate composed of the OSK-1peptide conjugated to a mouse IL-17A epitope peptide (SEQ ID NO: 40) viaan ε-Acp linker.

FIG. 13 shows the antibody production-inducing effect of OSK-1-IL-17Aconjugates.

FIG. 14 shows the results of the assessment of the medical efficacy ofOSK-1-IL-17A conjugates based on the skin conditions of model mice ofimiquimod-induced psoriasiform dermatitis.

FIG. 15 shows the results of the assessment of the medical efficacy ofthe OSK-1-IL-17A conjugates based on the ear thickness of model mice ofimiquimod-induced psoriasiform dermatitis.

FIG. 16 shows the skin conditions of model mice of imiquimod-inducedpsoriasiform dermatitis on Day 6 after the start of psoriasis inductionpreceded by vaccination with an OSK-1-IL-17A conjugate or a KLH-IL-17Aconjugate.

FIG. 17 shows the comparison of the medical efficacies of theOSK-1-IL-17A conjugate and the KLH-IL-17A conjugate based on the skinconditions of model mice of imiquimod-induced psoriasiform dermatitis.

FIG. 18 shows the comparison of the medical efficacies of theOSK-1-IL-17A conjugate and the KLH-IL-17A conjugate based on the earthickness of model mice of imiquimod-induced psoriasiform dermatitis.

FIG. 19 shows the results of involucrin staining of the skin of modelmice of imiquimod-induced psoriasiform dermatitis on Day 12 after thestart of psoriasis induction preceded by vaccination with theOSK-1-IL-17A conjugate or the KLH-IL-17A conjugate.

FIG. 20 shows the results of F4/80 staining of the skin of model mice ofimiquimod-induced psoriasiform dermatitis on Day 12 after the start ofpsoriasis induction preceded by vaccination with the OSK-1-IL-17Aconjugate or the KLH-IL-17A conjugate.

FIG. 21 shows the results of Gr-1 staining of the skin of model mice ofimiquimod-induced psoriasiform dermatitis on Day 12 after the start ofpsoriasis induction preceded by vaccination with the OSK-1-IL-17Aconjugate or the KLH-IL-17A conjugate.

FIG. 22 shows the blood IL-17 concentration measured in model mice ofimiquimod-induced psoriasiform dermatitis which had undergonevaccination with the OSK-1-IL-17A conjugate or the KLH-IL-17A conjugateprior to psoriasis induction.

FIG. 23 shows the results of the assessment of the medical efficacy ofan OSK-1-PCSK9 conjugate based on the blood PCSK9 concentration.

DESCRIPTION OF EMBODIMENTS

The present invention provides a conjugate vaccine targeting adisease-causing biological protein. The vaccine of the present inventioncontains a complex of a peptide consisting of an amino acid sequencethat is the same or substantially the same as the amino acid sequence ofSEQ ID NO: 1 (ELKLIFLHRLKRLRKRLKRK) and an epitope of thedisease-causing biological protein. As used herein, the peptideconsisting of the amino acid sequence of SEQ ID NO: 1 may be referred toas an OSK-1 peptide or OSK-1. In addition, as used herein, the terms“conjugate” and “complex” are interchangeably used.

As used herein, the term “vaccine” means a composition containing animmunogen which elicits immunoreaction after administered to an animal.Therefore, stated in another way, the vaccine of the present inventionis an immunogenic composition. The vaccine of the present invention isnot limited to a vaccine to be administered for preventive purposes, andmay be a vaccine to be administered for therapeutic purposes afterdisease onset.

The amino acid sequence that is substantially the same as the amino acidsequence of SEQ ID NO: 1 is, for example, an amino acid sequence that isthe same as the amino acid sequence of SEQ ID NO: 1 except for 1 to 4amino acid deletions, substitutions or additions. Preferred is an aminoacid sequence that is the same as the amino acid sequence of SEQ ID NO:1 except for deletions, substitutions or additions of 1 to 3 aminoacids, more preferably 1 or 2 amino acids, still more preferably oneamino acid. The peptide consisting of an amino acid sequence that issubstantially the same as the amino acid sequence of SEQ ID NO: 1 ispreferably a peptide having an activity that is substantially the sameas that of the peptide consisting of the amino acid sequence of SEQ IDNO: 1. Specifically, a complex of this peptide and an epitope of abiological protein has an antibody production-inducing capabilitycomparable (for example, about 0.5- to 2-fold) to that of a complex ofthe peptide consisting of the amino acid sequence of SEQ ID NO: 1 andthe same epitope of the biological protein.

The disease-causing biological protein is not particularly limited, andexamples include DPP4, IL-17A, IgE, S100A9 and PCSK9. As used herein,the term “biological protein” means an endogenous protein.

DPP4 (dipeptidyl peptidase-4) is an enzyme that degrades incretin, whichis involved in insulin secretion. Incretin is a gastrointestinal hormonethat promotes insulin secretion during hyperglycemia such aspostprandial hyperglycemia to reduce the blood glucose level. Therefore,the inhibition of the function of DPP4 for preventing the degradation ofincretin increases incretin concentration, thereby promoting insulinsecretion and thus improving the symptoms of diabetes mellitus. Based onthis mechanism, many DPP4 inhibitors have been developed and approved asantidiabetic drugs. Therefore, vaccines capable of inducing an anti-DPP4antibody are expected to treat diabetes mellitus.

The epitope of human DPP4 used for an OSK-1-DPP4 conjugate is notparticularly limited as long as it is an epitope capable of inducing theproduction of an antibody that inhibits the functions of DPP4(neutralizing antibody). Preferable examples of the epitope includepeptides consisting of any of the following amino acid sequences.

(SEQ ID NO: 2) ENSTFLEFG (SEQ ID NO: 3) NKRQLTTFE (SEQ ID NO: 4)KNTYRLKLYS (SEQ ID NO: 5) YSDESLOYPK (SEQ ID NO: 6) PPHFDKEKEY(SEQ ID NO: 7) GLPTPEDNLD (SEQ ID NO: 8) FSKEAKYYQ (SEQ ID NO: 9)NSSVYLENSTFLEFG

The accession numbers of the amino acid sequence of human DPP4 and thenucleotide sequence of the gene encoding human DPP4 are NP_001926 (NCBI)and NM_001935 (NCBI), respectively.

Peptides which consist of an amino acid sequence that is the same as theamino acid sequence of any of SEQ ID NOs: 2 to 9 except for 1 or 2 aminoacid deletions, substitutions or additions and which are capable ofinducing the production of an antibody that inhibits the functions ofDPP4 are also preferable as the epitope of human DPP4 used for theOSK-1-DPP4 conjugate.

IL-17A (interleukin-17A) is a homodimeric glycoprotein and is alsoreferred to simply as IL-17. IL-17A, which is mainly produced byactivated T cells, acts on a wide range of cells such as fibroblasts,epithelial cells, vascular endothelial cells and macrophages to recruitvarious factors such as inflammatory cytokines, chemokines and celladhesion factors, thereby inducing inflammation. Secukinumab, a humananti-human IL-17A monoclonal antibody, is used as a therapeutic drug forpsoriasis vulgaris and psoriasis arthropathica. In addition, IL-17A isreportedly involved in autoimmune diseases such as rheumatoid arthritis,multiple sclerosis and inflammatory bowel disorder, various cancersincluding non-small-cell lung cancer, colorectal cancer and pancreaticcancer, arteriosclerosis, etc. Therefore, vaccines capable of inducingan anti-IL-17A antibody are expected to treat these IL-17A-associateddiseases.

The epitope of human IL-17A used for an OSK-1-IL-17A conjugate is notparticularly limited as long as it is an epitope capable of inducing theproduction of an antibody that inhibits the functions of IL-17A(neutralizing antibody). Preferable examples of the epitope includepeptides consisting of any of the following amino acid sequences.

(SEQ ID NO: 10) RSSDYYNR (SEQ ID NO: 11) PKRSSDYYNRSTSPW (SEQ ID NO: 29)CPNSEDKNFPR (SEQ ID NO: 30) RNEDPERYPS (SEQ ID NO: 31) NRSTSPW(SEQ ID NO: 32) LHRNEDP (SEQ ID NO: 33) RYPSVIWEA (SEQ ID NO: 34)PKRSSDYYNR (SEQ ID NO: 35) INPKRSSDYYNR (SEQ ID NO: 36) ININPKRSSDYYNR

The accession numbers of the amino acid sequence of human IL-17A and thenucleotide sequence of the gene encoding human IL-17A are NP_002181(NCBI) and NM_002190 (NCBI), respectively.

Peptides which consist of an amino acid sequence that is the same as theamino acid sequence of any of SEQ ID NOs: 10, 11 and 29 to 36 except for1 or 2 amino acid deletions, substitutions or additions and which arecapable of inducing the production of an antibody that inhibits thefunctions of IL-17A are also preferable as the epitope of human IL-17Aused for the OSK-1-IL-17A conjugate. Moreover, a peptide consisting of apartial sequence containing the amino acid sequence of residues 3 to 10of SEQ ID NO: 11 is also preferable as the epitope of human IL-17A usedfor the OSK-1-IL-17A conjugate.

Allergy is caused by specific substances (allergens), and after anallergen enters the body, the immune system responds to the allergen andinduces IgE production. The produced IgE antibodies are attached to mastcells and basophils and maintained on the surface of these cells. Whenthe same allergen enters the body again, IgE and the allergen bindtogether, thereby inducing the release of chemical mediators such ashistamine and leukotriene, which trigger allergic symptoms. In the casewhere an allergy to a specific allergen has developed, overproduction ofspecific IgE against the allergen deteriorates allergic symptoms.Allergic symptoms can be alleviated by inhibiting or abolishing theactions of IgE, and for example, the anti-human IgE antibody omalizumabis used for the treatment of bronchial asthma. Therefore, vaccinescapable of inducing an anti-IgE antibody are expected to treat allergicdiseases such as bronchial asthma, pollen allergy, allergicconjunctivitis and atopic dermatitis.

The epitope of human IgE used for an OSK-1-IgE conjugate is notparticularly limited as long as it is an epitope capable of inducing theproduction of an antibody that inhibits the functions of IgE(neutralizing antibody). A preferable example of the epitope is apeptide consisting of the following amino acid sequence.

YQCRVTHPHLP (SEQ ID NO: 12)

The accession numbers of the amino acid sequence of the constant regionof a human immunoglobulin e chain and the nucleotide sequence of thegene encoding the constant region of a human immunoglobulin a chain areP01854 (UniProtKB) and NG_001019 (NCBI), respectively.

Peptides which consist of an amino acid sequence that is the same as theamino acid sequence of SEQ ID NO: 12 except for 1 or 2 amino aciddeletions, substitutions or additions and which are capable of inducingthe production of an antibody that inhibits the functions of IgE arealso preferable as the epitope of human IgE used for the OSK-1-IgEconjugate.

S100 proteins are a family of calcium-binding proteins with two EF-handdomains and are cell type-specifically expressed. So far, 20 subfamilieshave been identified. S100A9 (also referred to as MRP14) is alow-molecular-weight calcium-binding S100 protein and is known to beinvolved in various inflammatory diseases. In addition, elevated levelsof serum S100A9 are observed in patients of many inflammatory diseasesincluding giant cell arteritis, cystic fibrosis, rheumatoid arthritis,dermatosis, chronic inflammatory bowel disease, chronic bronchitis, somemalignant tumors and autoimmune disease. Moreover, blocking of S100A9reportedly could prevent thrombus formation without increasing the riskof hemorrhage. Therefore, vaccines capable of inducing an anti-S100A9antibody are expected to prevent thrombus formation in atherothrombosis,myocardial infarction and cerebral infarction.

The epitope of human S100A9 used for an OSK-1-S100A9 conjugate is notparticularly limited as long as it is an epitope capable of inducing theproduction of an antibody that inhibits the functions of S100A9(neutralizing antibody). A preferable example of the epitope is apeptide consisting of the following amino acid sequence.

GHHHKPGLGE (SEQ ID NO: 13)

The accession numbers of the amino acid sequence of human S100A9 and thenucleotide sequence of the gene encoding human S100A9 are NP_002956(NCBI) and NM_002965 (NCBI), respectively.

Peptides which consist of an amino acid sequence that is the same as theamino acid sequence of SEQ ID NO: 13 except for 1 or 2 amino aciddeletions, substitutions or additions and which are capable of inducingthe production of an antibody that inhibits the functions of S100A9 arealso preferable as the epitope of human S100A9 used for the OSK-1-S100A9conjugate.

PCSK9 (proprotein convertase subtilisin/kexin type 9) was identified asa causative gene of familial hypercholesterolemia. Subsequent studiesconfirmed that PCSK9 degrades LDL receptors expressed on the surface ofhepatocytes and reduces the ability of the liver to take up LDL-C fromthe blood. By inhibiting the functions of PCSK9, the degradation of LDLreceptors can be prevented and the uptake of blood cholesterol into theliver can be facilitated, thus enabling the reduction of the bloodcholesterol level. For example, the anti-human PCSK9 monoclonal antibodyevolocumab is used for the treatment of familial hypercholesterolemiaand hypercholesterolemia, and RNAi drugs that inhibit the functions ofPCSK9 have been developed. Therefore, vaccines capable of inducing ananti-PCSK9 antibody are expected to treat hypercholesterolemia.

The epitope of human PCSK9 used for an OSK-1-PCSK9 conjugate is notparticularly limited as long as it is an epitope capable of inducing theproduction of an antibody that inhibits the functions of PCSK9(neutralizing antibody). Preferable examples of the epitope includepeptides consisting of any of the following amino acid sequences.

(SEQ ID NO: 14) LRPRGQPNQC (SEQ ID NO: 15) SRHLAQASQ (SEQ ID NO: 16)SRSGKRRGER

The accession numbers of the amino acid sequence of human PCSK9 and thenucleotide sequence of the gene encoding human PCSK9 are NP_777596(NCBI) and NM_174936 (NCBI), respectively.

Peptides which consist of an amino acid sequence that is the same as theamino acid sequence of any of SEQ ID NOs: 14 to 16 except for 1 or 2amino acid deletions, substitutions or additions and which are capableof inducing the production of an antibody that inhibits the functions ofPCSK9 are also preferable as the epitope of human PCSK9 used for theOSK-1-PCSK9 conjugate.

In the case where experiments using laboratory animals (mice etc.) areperformed to examine the effect of the vaccine of the present invention,it is preferable to use an epitope sequence of the laboratory animalcorresponding to the above-mentioned human epitope sequence. The epitopesequence of the laboratory animal to be used can be designed byalignment of the amino acid sequence of a target protein obtained fromknown databases (NCBI etc.) with the corresponding human amino acidsequence. Table 1 lists the mouse epitope sequences corresponding to theabove-mentioned human epitope sequences of SEQ ID NOs: 2 to 16.

TABLE 1 Positions Positions in in full-length full-length Targetamino acid amino acid protein Mouse epitope sequence sequenceHuman epitope sequence sequence DPP4 ENSTFESFG (SEQ ID NO: 17) 89-97ENSTFDEFG (SEQ ID NO: 2) 91-99 NKRQLITEE (SEQ ID NO: 3) 132-140NKRQLITEE (SEQ ID NO: 3) 138-146 KSTFRVKSYS (SEQ ID NO: 18) 48-57KNTYRLKLYS (SEQ ID NO: 4) 50-59 YSDESLQYPK (SEQ ID NO: 5) 235-244YSDESLQYPK (SEQ ID NO: 5) 241-250 PPHFDKSKKY (SEQ ID NO: 6) 525-534PPHFDKSKKY (SEQ ID NO: 6) 531-540 GLPPEDNLD (SEQ ID NO: 19) 666-675GLPTPEDNLD (SEQ ID NO: 7) 672-681 FSKEAKYYQ (SEQ ID NO: 8) 455-463FSKEAKYY (SEQ ID NO: 8) 461-469 NSSIFLENSTFESFG (SEQ ID NO: 20) 83-97NSSVFLENSTFDEFG (SEQ ID NO: 9) 85-99 IL-17A RPSDYLNR (SEQ ID NO: 21)65-72 RSSDYYNR (SEQ ID NO: 10) 62-69 SRRPSDYLNRSTSPW (SEQ ID NO: 22)63-77 PKRSSDYYNRSTSPW (SEQ ID NO: 11) 60-74 CPNTEAKDFLQ (SEQ ID NO: 37)35-45 CPNSEDKNFPR (SEQ ID NO: 29) 33-43 RNEDPDRYPS (SEQ ID NO: 38) 81-90RNEDPERYPS (SEQ ID NO: 30) 78-87 NRSTSPW (SEQ ID NO: 31) 71-77NRSTSPW (SEQ ID NO: 31) 68-74 LHRNEDP (SEQ ID NO: 32) 79-85LHRNEDP (SEQ ID NO: 32) 76-82 YPSVIWEA (SEQ ID NO: 33) 87-95RYPSVIWEA (SEQ ID NO: 33) 84-92 RYPSDYLNR (SEQ ID NO: 39) 63-72PKRSSDYYNR (SEQ ID NO: 34) 60-69 VSSRRPSDYLNR (SEQ ID NO: 40) 61-72TNPKRSSDYYNR (SEQ ID NO: 35) 58-69 AKVSSRRPSDYLNR (SEQ ID NO: 41) 59-72TNTNPKRSSDYYNR (SEQ ID NO: 36) 56-69 IgE YQCIVDHPDFP (SEQ ID NO: 23)283-293 YQCRVTHPHLP (SEQ ID NO: 12) 297-307 PCSK9PALRSRRQPG (SEQ ID NO: 25) 580-589 LRPRGQPNQC (SEQ ID NO: 14) 580-589CRSRPSAKA (SEQ ID NO: 26) 682-690 SRHLAQASQ (SEQ ID NO: 15) 682-690SFSRSGRRRG (SEQ ID NO: 27) 491-500 SRSGKRRGER (SEQ ID NO: 16) 491-500S100A9 GHSHGKGCGK (SEQ ID NO: 24) 104-113 GHHHKPGLGE (SEQ ID NO: 13)102-111

In Table 1, the full-length amino acid sequence of human DPP4 is basedon the amino acid sequence registered with the accession numberNP_001926, the full-length amino acid sequence of mouse DPP4 is based onthe amino acid sequence registered with the accession number AAH22183,the full-length amino acid sequence of human IL-17A is based on theamino acid sequence registered with the accession number NP_002181, thefull-length amino acid sequence of mouse IL-17A is based on the aminoacid sequence registered with the accession number NP_034682, thefull-length amino acid sequence of human IgE is based on the amino acidsequence registered with the accession number P01854, the full-lengthamino acid sequence of mouse IgE is based on the amino acid sequenceregistered with the accession number P06336, the full-length amino acidsequence of human S100A9 is based on the amino acid sequence registeredwith the accession number NP_002956, the full-length amino acid sequenceof mouse S100A9 is based on the amino acid sequence registered with theaccession number CAC14292, the full-length amino acid sequence of humanPCSK9 is based on the amino acid sequence registered with the accessionnumber NP_777596, and the full-length amino acid sequence of mouse PCSK9is based on the amino acid sequence registered with the accession numberNP_705793.

The length of the epitope of the biological protein used for the vaccineof the present invention is not particularly limited, but the length ispreferably 20 amino acids or less, more preferably 15 amino acids orless, still more preferably 13 amino acids or less, yet still morepreferably 12 amino acids or less, yet still more preferably 11 aminoacids or less, and yet still more preferably 10 amino acids or less. Theminimum of the length is not particularly limited, but the length ispreferably 3 amino acids or more, more preferably 4 amino acids or more,still more preferably 5 amino acids or more, and still more preferably 6amino acids or more.

In the complex of the OSK-1 peptide and the epitope of the biologicalprotein, the OSK-1 peptide and the epitope may be conjugated directly orvia a linker (used as a synonym for a spacer). The linker is notparticularly limited as long as it is capable of connecting the OSK-1peptide with the epitope of the biological protein. Examples of thelinker include aminocarboxylic acids such as β-aminoalanine,γ-aminobutyric acid, ε-aminocaproic acid, 7-aminoheptanoic acid,12-aminolauric acid, glutamic acid and p-aminobenzoic acid. Otherexamples include L-amino acids, which are present in naturally occurringproteins, and D-isomers thereof. In Examples of the presentspecification, ε-aminocaproic acid is used, but is a non-limitingexample.

The sequential order of the OSK-1 peptide and the epitope of thebiological protein in the complex is not particularly limited. Thecomplex may have the OSK-1 peptide at the N-terminal side and theepitope of the biological protein at the C-terminal side. Also, thecomplex may have the epitope of the biological protein at the N-terminalside and the OSK-1 peptide at the C-terminal side. Preferably, thecomplex has the OSK-1 peptide at the N-terminal side and the epitope ofthe biological protein at the C-terminal side.

In the complex, the amino acid at the N-terminus is preferablyacetylated. In the complex, the amino acid at the C-terminus ispreferably amidated. More preferably, the amino acid at the N-terminusof the complex is acetylated, and the amino acid at the C-terminus ofthe complex is amidated.

The complex of the OSK-1 peptide and the epitope of the biologicalprotein can be prepared as a fusion protein. For example, the complexcan be prepared by using known genetic engineering techniques,specifically by preparing a fusion gene encoding a fusion protein of theOSK-1 peptide and the epitope of the biological protein, constructing arecombinant expression vector having the fusion gene expressiblyinserted, transfecting the recombinant expression vector intoappropriate host cells for expression of a recombinant protein, andpurifying the recombinant protein. Alternatively, the preparation of thecomplex of the OSK-1 peptide and the epitope of the biological proteincan be performed using the above-described fusion gene in combinationwith a known in vitro coupled transcription-translation system (forexample, a cell-free protein synthesis system derived from rabbitreticulocytes, wheat germ or Escherichia coli). Moreover, in the casewhere a bacteriophage is used to provide the expression of the complexof the OSK-1 peptide and the epitope of the biological protein as arecombinant protein on the surface of the bacteriophage, the complexexpressed on the phage surface may be directly administered to a targetanimal without isolation or purification.

The complex of the OSK-1 peptide and the epitope of the biologicalprotein can be produced by a solid phase synthesis method (e.g., theFmoc method and the Boc method) or a liquid phase synthesis methodaccording to a known ordinary peptide synthesis protocol. In the casewhere the complex has the OSK-1 peptide directly conjugated to theepitope of the biological protein, or in the case where the complex hasthe OSK-1 peptide conjugated to the epitope of the biological proteinvia an appropriate amino acid(s), the whole complex can be synthesized.Alternatively, the OSK-1 peptide and the epitope of the biologicalprotein may be synthesized separately before coupling of the twopeptides via an appropriate linker.

The linker can be introduced by a well-known method used in peptidesynthesis chemistry. Firstly, a peptide fragment whose N-terminal end issupposed to be coupled to a linker is produced by a standard method.Then, an aminocarboxylic acid as the linker, in which the amino group isappropriately protected, and the peptide fragment are condensed instandard conditions using a condensing agent used in peptide synthesischemistry. After removal of the protective group for the linker group,the desired amino acid is introduced at the next position. An example ofsuch a method is the solid phase peptide synthesis method often employedin a peptide automatic synthesizer. Examples of the protective group forthe aminocarboxylic acid used in such a procedure include a tBOC(tert-butyl oxy carbonyl) group and an FMOC (fluorenyl-methoxy-carbonyl)group. Examples of the condensing agent include carbodiimide compoundssuch as DCC (N,N′-dicyclohexylcarbodiimide) and EDC(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide); imidazole compoundssuch as CDI (1,1′-carbonyldiimidazole); phosphonium salt compounds suchas BOP (benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate); and uronium salt compounds such as HBTU(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)and HCTU (O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate). Also, a desired conjugate can be obtained byanother procedure using an appropriate combination of the aboveconditions. Specifically, first, a specific peptide fragment of OSK-1and a peptide fragment of the epitope sequence of the target biologicalprotein are separately produced and appropriately protected. Next, thesefragments are condensed with an appropriately protected linker group andthen deprotected in appropriate conditions.

Without an adjuvant, the vaccine of the present invention can induce anantibody that inhibits the functions of the disease-causing biologicalprotein, and therefore is highly useful. However, the vaccine of thepresent invention may contain one or more adjuvants. In the case wherethe vaccine of the present invention contains an adjuvant, the adjuvantcan be selected as appropriate from known adjuvants. Specific examplesof the known adjuvants include aluminum adjuvants (for example, aluminumsalts such as aluminum hydroxide, aluminum phosphate and aluminumsulfate, or any combination thereof), complete or incomplete Freund'sadjuvant, TLR ligands (for example, CpG, Poly(I:C), Pam3CSK4, etc.),BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin andformylmethionyl peptides. Preferable adjuvants are aluminum adjuvants,TLR ligands and a combination of these. In the case where the vaccine ofthe present invention contains an adjuvant, the amount of the adjuvantis not particularly limited and can be selected as appropriate accordingto the kind of the adjuvant etc. For example, in the case where analuminum adjuvant (aluminum hydroxide) and CpG are used in combination,it is preferable that the vaccine contains an about 1- to 100-foldamount of the aluminum adjuvant and an about 1- to 50-fold amount of CpGrelative to the amount of the fusion protein of the present invention ona mass basis.

The vaccine of the present invention can be administered orally orparenterally. Examples of the parenteral administration includeintraperitoneal administration, subcutaneous administration,intracutaneous administration, intramuscular administration, intravenousadministration, intranasal administration, transdermal administration,transmucosal administration, sublingual administration and inhalationadministration. Preferred is parenteral administration, and morepreferred are intracutaneous administration, subcutaneous administrationand intramuscular administration. For parenteral administration,microneedle injection, non-needle injection, a stamping method, etc. maybe employed.

For the formulation of the vaccine of the present invention, the complexof the OSK-1 peptide and the epitope of the biological protein, apharmaceutically acceptable carrier and if needed an additive areblended and formed into a dosage form. Specific examples of the dosageform include oral preparations such as tablets, coated tablets, pills,powders, granules, capsules, solutions, suspensions and emulsions; andparenteral preparations such as injections, infusions, suppositories,ointments and patches. The amount of the carrier or the additive to beused is determined as appropriate based on the range of amountconventionally used in the pharmaceutical field. The carrier or theadditive that can be used is not particularly limited, and examplesinclude various carriers such as water, physiological saline, otheraqueous solvents, and aqueous or oily bases; and various additives suchas excipients, binders, pH adjusters, disintegrants, absorptionenhancers, lubricants, colorants, corrigents and fragrances.

Examples of the additive used for solid oral preparations includeexcipients such as lactose, mannitol, glucose, microcrystallinecellulose and corn starch; binders such as hydroxypropyl cellulose,polyvinylpyrrolidone and magnesium aluminometasilicate; dispersants suchas corn starch; disintegrants such as calcium carboxymethyl cellulose;lubricants such as magnesium stearate; solubilizing agents such asglutamic acid and aspartic acid; stabilizers; water soluble polymersincluding celluloses such as hydroxypropyl cellulose,hydroxypropylmethyl cellulose and methyl cellulose, and syntheticpolymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinylalcohol; sweeteners such as white sugar, powder sugar, sucrose,fructose, glucose, lactose, reduced malt sugar syrup (maltitol syrup),reduced malt sugar syrup powder (maltitol syrup powder), high-glucosecorn syrup, high-fructose corn syrup, honey, sorbitol, maltitol,mannitol, xylitol, erythritol, aspartame, saccharin and saccharinsodium; and coating agents such as white sugar, gelatin, hydroxypropylcellulose and hydroxypropylmethyl cellulose phthalate.

The formulation of liquid oral preparations involves dissolution,suspension or emulsification in a generally used diluent. Examples ofthe diluent include purified water, ethanol and a mixture thereof. Theoral liquid preparations may further contain a wetting agent, asuspending agent, an emulsifier, a sweetener, a flavoring agent, afragrance, a preservative, a buffering agent and/or the like.

Examples of the additive used for injections for parenteraladministration include isotonizing agents such as sodium chloride,potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax,glucose and propylene glycol; buffering agents such as a phosphatebuffer solution, an acetate buffer solution, a borate buffer solution, acarbonate buffer solution, a citrate buffer solution, a Tris buffersolution, a glutamate buffer solution and an ε-aminocaproate buffersolution; preservatives such as methyl parahydroxybenzoate, ethylparahydroxybenzoate, propyl parahydroxybenzoate, butylparahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkoniumchloride, sodium dehydroacetate, disodium edetate, boric acid and borax;thickeners such as hydroxyethyl cellulose, hydroxypropyl cellulose,polyvinyl alcohol and polyethylene glycol; stabilizers such as sodiumhydrogen sulfite, sodium thiosulfate, disodium edetate, sodium citrate,ascorbic acid and dibutylhydroxytoluene; and pH adjusters such ashydrochloric acid, sodium hydroxide, phosphoric acid and acetic acid.The injection may further contain an appropriate solubilizer. Examplesof the solubilizer include alcohols such as ethanol; polyalcohols suchas propylene glycol and polyethylene glycol; and nonionic surfactantssuch as polysorbate 80, polyoxyethylene hydrogenated castor oil 50,lysolecithin and Pluronic polyol. Liquid preparations such as injectionscan be preserved in a frozen state, or in a dried state after waterremoval by lyophilization etc. Lyophilized preparations can bereconstituted in distilled water for injection or the like just beforeuse.

The vaccine of the present invention can be administered to any animal(a human or a non-human animal) with an immune system. Examples of theanimal include mammals such as humans, monkeys, cattle, horses, pigs,sheep, goats, dogs, cats, guinea pigs, rats and mice; and birds such aschickens, ducks and geese. The vaccine of the present invention isuseful as a veterinary drug, but is preferably used for human childrenand human adults.

In the administration of the vaccine of the present invention, thedosing frequency and interval are not particularly limited. For example,the vaccine may be administered once, or multiple times at intervals ofabout two days to about eight weeks. The dose of the vaccine varies withthe administration subject, the administration method, etc., but thedose per administration is preferably about 0.01 μg to about 10 mg, morepreferably about 0.1 μg to about 1 mg, and still more preferably about 1μg to about 0.1 mg.

The present invention includes a method for preventing or treating adisease caused by a target biological protein, the method comprisingadministering an effective amount of the vaccine of the presentinvention to an animal.

Further, the present invention includes the vaccine of the presentinvention for use in prevention or treatment of a disease caused by thebiological protein.

Moreover, the present invention includes use of the vaccine of thepresent invention for production of a medicament for prevention ortreatment of a disease caused by the biological protein.

OSK-1, which is used as the carrier protein for the epitope of thedisease-causing biological protein in the vaccine of the presentinvention, is a peptide of 20 amino acids with a very low antigenicity.Therefore, OSK-1 is less likely to cause unfavorable effects and sideeffects attributable to the carrier protein per se. In spite of itsshort length, OSK-1 has a strong adjuvant effect, and therefore iscapable of imparting the vaccine with an effective capability ofinducing antibody production. Moreover, the complex of OSK-1 and theepitope of the biological protein is capable of inducing the productionof not only Th2-type antibodies such as IgG1, but also a large amount ofTh1-type antibodies such as IgG2a, IgG2b and IgG3. Therefore, thecomplex is less likely to cause side effects such as allergic reactionsand is very useful. Furthermore, in the case where carrier proteins fromnatural source, such as KLH, are used, their impurities may post aproblem, but OSK-1 is free from such a problem because OSK-1 can beproduced by chemical synthesis. Furthermore, the short peptide OSK-1 isadvantageous in terms of reduced production cost.

EXAMPLES

Hereinafter, the present invention will be illustrated in detail byexamples, but the present invention is not limited thereto.

Production Example of Peptide

A protected peptide-bound resin was synthesized by the Fmoc method usinga fully-automatic solid-phase synthesizer according to the protocoldescribed in: Solid Phase Peptide Synthesis, Pierce (1984); Fmoc SolidSynthesis: A Practical Approach, Oxford University Press (2000); TheFifth Series of Experimental Chemistry (Jikken Kagaku Kouza), vol. 16,Synthesis of Organic Compounds IV; or the like. To the protectedpeptide-bound resin, trifluoroacetic acid (TFA) and a scavenger (amixture of thioanisole, ethanedithiol, phenol, triisopropylsilane, waterand the like) were added for cleavage of the protected peptide from theresin and deprotection thereof. Thus, the peptide of interest wasobtained as a crude product. For the purification of the peptide, thecrude product was applied to a reverse-phase HPLC column (ODS) andelution was performed with a gradient of 0.1% TFA-H₂O/CH₃CN. Thefractions containing the peptide of interest were combined andfreeze-dried, and the peptide of interest was obtained. The amino acidsequence of the synthesized peptide was confirmed with the amino acidsequencer G1000A (Hewlett Packard), PPSQ-23A (Shimadzu Corporation) orProcise cLC (ABI). The obtained peptide was subjected to N-terminalacetylation.

Example 1: Examination of Antibody Production Induced by OSK-1-DPP4Conjugate—Part 1

An OSK-1-DPP4 conjugate composed of an OSK-1 peptide (SEQ ID NO: 1)conjugated to a mouse DPP4 epitope peptide (SEQ ID NO: 17) via an ε-Acplinker (Ac-ELKLIFLHRLKRLRKRLKRK-X-ENSTFESFG (X=ε-Acp)) was assessed forthe antibody production-inducing effect. For this test, the followingtwo groups were prepared: a physiological saline group and an OSK-1-DPP4conjugate group (100 μg/mouse) (6 animals per group). Test samples wereintracutaneously administered to Balb/c mice 3 times at 2-weekintervals. Before the first administration and every 2 weeks until 8weeks after the first administration, blood samples were collected, andthe antibody titer against the DPP4 epitope peptide was measured byELISA.

The results are shown in FIG. 1. After the administration of theOSK-1-DPP4 conjugate, antibody production against the DPP4 epitopepeptide was observed.

Example 2: Examination of Antibody Production Induced by OSK-1-DPP4Conjugate—Part 2

The antibody production-inducing effect of the same OSK-1-DPP4 conjugateas used in Example 1 was compared with that of a conjugate of KLH(keyhole limpet hemocyanin) and the mouse DPP4 epitope peptide (SEQ IDNO: 17) (KLH-DPP4). For this test, the following three groups wereprepared: a KLH-DPP4 conjugate group (20 μg/mouse), an OSK-1-DPP4conjugate group (100 μg/mouse) and an OSK-1-DPP4 conjugate (100μg/mouse) plus Alum (Alhydrogel 2% (InvivoGen) 1 mg/mouse) group (2animals per group). Test samples were intracutaneously administered toBalb/c mice 3 times at 2-week intervals. Before the firstadministration, every 2 weeks until 6 weeks after the firstadministration, and 12 weeks after the first administration, bloodsamples were collected, and the antibody titer against the DPP4 epitopepeptide was measured by ELISA.

The results are shown in FIG. 2. The antibody production-inducingcapability of the OSK-1-DPP4 conjugate was comparable to that of theKLH-DPP4 conjugate. In addition, synergistic effect was observed in theOSK-1-DPP4 conjugate plus Alum group.

Example 3: Analysis of IgG Subclass of Produced Antibodies

The IgG subclass of antibodies produced by vaccination with the sameOSK-1-DPP4 conjugate as used in Examples 1 and 2 was compared with thatof antibodies produced by vaccination with the same KLH-DPP4 conjugateas used in Example 2. For this test, the following three groups wereprepared: an OSK-1-DPP4 conjugate group (100 μg/mouse), a KLH-DPP4conjugate (20 μg/mouse) plus Alum (Alhydrogel 2% (InvivoGen) 1 mg/mouse)group and a KLH-DPP4 conjugate (20 μg/mouse) plus OSK-1 (100 μg/mouse)group. Test samples were intracutaneously administered to Balb/c mice 3times at 2-week intervals and at 9 weeks after the start of theadministration, 4 times in total. At 11 weeks after the start of theadministration, blood samples were collected, and the titer of each IgGsubclass of antibodies against the DPP4 epitope peptide was measured byELISA.

The results are shown in FIG. 3. In the KLH-DPP4 conjugate plus Alumgroup, the titer of IgG1, which is a Th2-type IgG subclass, had atendency to be particularly higher than those of other IgG subclasses.In the OSK-1-DPP4 conjugate group, IgG2a and IgG2b, which are Th1-typeIgG subclasses, were also produced at high levels in addition to IgG1.Similarly, in the KLH-DPP4 conjugate plus OSK-1 group, the titers ofIgG2b and IgG3, which are Th1-type IgG subclasses, were high.

Reference Example 1: Examination of Antitumor Effect of OSK-1-WT1Conjugate—Part 1

An OSK-1-WT1 conjugate composed of the OSK-1 peptide (SEQ ID NO: 1)conjugated to a WT1 peptide (SEQ ID NO: 28) via an ε-Acp linker(Ac-ELKLIFLHRLKRLRKRLKRK-X-RMFPNAPYL (X=ε-Acp)) was assessed for theantitumor effect. In addition, the adjuvant effect of the OSK-1 peptideon the antitumor effect of a WT1 peptide vaccine was also examined. Forthis test, the following four groups were prepared: a physiologicalsaline group, a WT1 peptide (100 μg/mouse) plus poly(I:C) (InvivoGen;Poly(I:C) HMWVacciGrade, 50 g/mouse) group, aWT1 peptide (100 μg/mouse)plus OSK-1 peptide (100 μg/mouse) group and an OSK-1-WT1 conjugate (350μg/mouse) group (6 animals per group). The vaccines were administered toC57BL/6J mice once a week, 4 times in total. One week after the 4thadministration, B16F10 melanoma cells (1×10⁵ cells/mouse) were implantedinto the dorsal skin. The vaccines were administered again one weekafter the cell implantation. The body weight and the diameter of thetumor were measured twice a week after the cell implantation. Thediameter of the tumor was measured using a digital caliper, and thetumor volume was calculated by the formula: (major axis)×(minor axis)².

The results of the tumor volume are shown in FIG. 4, and the results ofthe survival rate are shown in FIG. 5. In the group where the WT1peptide vaccine was administered in combination with the OSK-1 peptideas the adjuvant, neither the inhibitory effect on tumor growth nor thesurvival benefit was observed. In contrast, in the OSK-1-WT1 conjugategroup, the inhibitory effect on tumor growth and the survival benefitwere observed.

Reference Example 2: Examination of Antitumor Effect of OSK-1-WT1Conjugate—Part 2

The antitumor effect of the same OSK-1-WT1 conjugate as used inReference Example 1 was assessed. For this test, the following fourgroups were prepared: a physiological saline group, a WT1 peptide (100μg/mouse) plus poly(I:C) (InvivoGen; Poly (I:C) HMW VacciGrade, 50μg/mouse) group, an OSK-1-WT1 conjugate (350 μg/mouse) group and acisplatin (5 mg/kg) group (10 animals per group). B16F10 melanoma cells(3×10⁵ cells/mouse) were implanted to C57BL/6J mice, and at the sameday, the vaccines were intracutaneously administered into the dorsalskin. The day of cell implantation was designated as Day 0. The vaccineswere administered on Day 7, Day 14, Day 21, Day 28 and Day 35. Twice aweek after the cell implantation, the diameter of the tumor was measuredusing a digital caliper, and the tumor volume was calculated by theformula: (major axis)×(minor axis)².

The results of the tumor volume are shown in FIG. 6, and the results ofthe survival rate are shown in FIG. 7. In the case where vaccinationstarted on the day of cell implantation, the inhibitory effect of theOSK-1-WT1 conjugate on tumor growth was modest, but the tumor volume waskept at lower levels than that of the physiological salineadministration group (negative control) until Day 32. In addition, thesurvival benefit in the OSK-1-WT1 conjugate group was comparable tothose in the WT1 peptide plus poly(I:C) group and the cisplatin group.

Example 4: Examination of Antibody Production Induced by OSK-1-DPP4Conjugates—Part 3

Seven types of mouse DPP4 epitope peptides (SEQ ID NOs: 3, 5, 6, 8, 18,19 and 20) and one type of human DPP4 epitope peptide (SEQ ID NO: 9)were separately conjugated to the OSK-1 peptide (SEQ ID NO: 1) via anε-Acp linker to produce 8 types of OSK-1-DPP4 conjugates. Hereinafter,each OSK-1-DPP4 conjugate is referred to as “OSK1-DDP4-sequence IDnumber”.

Six types of OSK-1-DPP4 conjugates containing different mouse DPP4epitope peptides (SEQ ID NOs: 3, 5, 6, 8, 18 and 19) wereintracutaneously administered to Balb/c mice at a dose of 100 μg peranimal 3 times at 2-week intervals. Before the first administration andevery 2 weeks after the first administration, blood samples werecollected, and the antibody titer against each epitope peptide wasmeasured by ELISA.

The antibody titers measured 4 weeks after the first administration areshown in Table 2. The antibody titer is expressed as a half maximumvalue. The half maximum value is a value of a dilution factor at whichthe absorbance of the serum sample is half of the maximum absorbancemeasured in a measurement device. The half maximum value can be obtainedfrom a sigmoid curve created by plotting serum dilution factors againstmeasured absorbances.

TABLE 2 DPP4 vaccine Half Maximum ID Dose (μg) Half Maximum(4 W)OSK1-DPP4-3 100 <10 OSK1-DPP4-18 100 <10 OSK1-DPP4-5 100 20.8OSK1-DPP4-6 100 248.3 OSK1-DPP4-19 100 97.7 OSK1-DPP4-8 100 <10

Three types of OSK-1-DPP4 conjugates, OSK1-DDP4-6, OSK1-DDP4-9 andOSK1-DDP4-12, were intracutaneously administered to Balb/c mice at adose of 250 μg per animal 3 times at 2-week intervals. At 4, 9 and 13weeks after the first administration, blood samples were collected, andthe antibody titer against each epitope peptide was measured by ELISA.

The results are shown in Table 3. The antibody titer is expressed as ahalf maximum value.

TABLE 3 DPP4 vaccine Half Maximum 2 ID Dose (μg) 4 w 9 w 13 wOSK1-DPP4-6 250 329.4 280.3 408.6 OSK1-DPP4-9 250 <10 <10 53.6OSK1-DPP4-20 250 <10 10.4 44.9

Example 5: Assessment of OSK-1-DPP4 Conjugate by HbA1c Level Measurement

An OSK-1-DPP4 conjugate composed of the OSK-1 peptide (SEQ ID NO: 1)conjugated to the DPP4 epitope peptide (SEQ ID NO: 6) via an ε-Acplinker was intracutaneously administered at a dose of 100 μg per animal3 times at 2-week intervals to Balb/c mice which had been given freeaccess to a high fat diet (DIO SERIES DIET D12492, CLEA Japan). At 57days after the first administration, blood samples were collected, andas quickly as possible, each of the blood samples was partly transferredto a separate EDTA-containing tube. Each tube was inverted for mixing,and EDTA-treated blood samples were prepared. About 20 μL of eachEDTA-treated blood sample was transferred to a microtube for HbA1cmeasurement, and the HbA1c level was measured using Quo-Lab HbA1cAnalyzer (Nipro). The results show that the HbA1c level (%, NGSP) of theOSK-1-DPP4 conjugate group was improved (reduced by 0.4%) as comparedwith that of the control group (KLH administration group).

Example 6: Assessment of OSK-1-DPP4 Conjugates by Oral Glucose ToleranceTest (OGTT)

Two types of OSK-1-DPP4 conjugates composed of the OSK-1 peptide (SEQ IDNO: 1) conjugated to the DPP4 epitope peptide (SEQ ID NO: 6 or 9) via anε-Acp linker were intracutaneously administered at a dose of 250 μg peranimal 3 times at 2-week intervals to Balb/c mice which had been givenfree access to a high fat diet (DIO SERIES DIET D12492, CLEA Japan). TheOSK-1-DPP4 conjugates were further intracutaneously administered at adose of 250 μg per animal at 63 days after the first administration. Anoral glucose tolerance test (OGTT) was performed at 77 days after thefirst administration. The mice were under fasting conditions for about16 hours before the OGTT test, and during the OGTT test, the mice werestill under fasting conditions. A 20% glucose solution was orallyadministered at a single dose of 5 mL/kg to the mice. Blood collectionand blood glucose level measurement were performed under unanesthetizedconditions. The mouse tail was gently cut with a razor blade, and theglucose concentration in the blood leaking from the cut was measuredwith a blood glucose monitoring system (Nipro, FreeStyle Freedom). Theblood glucose levels were measured before glucose loading and at 30, 60,90 and 120 minutes after glucose loading, at 5 time points in total. Theblood glucose levels of each group were plotted against time, and theblood glucose area under the curve (glucose AUC) (time*mg/dl) wascalculated. The results show that the AUCs of both the OSK-1-DPP4conjugate administration groups were lower than that of the controlgroup (KLH administration group).

Example 7: Examination of IgE Production Induced by OSK-1-DPP4 Conjugate

In terms of IgE production against a target protein, OSK1-DPP4-17 wascompared with KLH-DPP4-17, which used KLH as a carrier protein insteadof OSK1. To a KLH-DPP4-17 group of mice, KLH-DPP4-17 and Alum(Alhydrogel 2% (InvivoGen)) were intracutaneously administered as amixture at doses of 20 μg/mouse and 1 mg/mouse, respectively. To anOSK-1-DPP4-17 group of mice, OSK-1-DPP4-17 was intracutaneouslyadministered at a dose of 100 μg/mouse. In either group, the conjugatewas intracutaneously administered to Balb/c mice 3 times at 2-weekintervals, and the 4th administration was performed at 11 weeks afterthe first administration. At 21 weeks after the first administration,blood samples were collected, and the amount of serum IgE antibodyagainst DPP4 was measured by ELISA.

The results are shown in FIG. 8. IgE production against the targetprotein was observed in the KLH-DPP4-17 group, but no IgE productionagainst the target protein was observed in the OSK1-DPP4-17 group.

Example 8: Examination of Antibody Production Induced by OSK-1-IL-17AConjugates (1) Production of OSK-1-IL-17A Conjugates

Ten types of mouse IL-17A epitope peptides (SEQ ID NOs: 21, 22, 31 to 33and 37 to 41) were separately conjugated to the OSK-1 peptide (SEQ IDNO: 1) via an ε-Acp linker to produce 10 types of OSK-1-IL-17Aconjugates. Hereinafter, each OSK-1-IL-17A conjugate is referred to as“OSK1-IL-sequence ID number”. FIGS. 9 to 12 show (A) the results ofamino acid analysis and (B) the results of HPLC analysis ofrepresentative 4 types of conjugates containing different epitopepeptides (SEQ ID NOs: 21, 31, 32 and 40). The results of the analyses ofthe other conjugates were substantially the same as these results.

(2) Measurement of Antibody Titer

Ten types of OSK-1-IL-17A conjugates were intracutaneously administeredto Balb/c mice at the doses indicated in Table 4 three times at 2-weekintervals. Before the first administration and every 2 weeks until 6weeks after the first administration, blood samples were collected, andthe antibody titer against each epitope peptide was measured by ELISA.

The results obtained 6 weeks after the first administration are shown inTable 4. The antibody titers measured 2, 4 and 6 weeks after the firstadministration are shown in FIG. 13. The antibody titer is expressed asa half maximum value.

TABLE 4 IL-17 vaccine Half Maximum ID Dose Half Maximum(6 W) OSK1-IL-21300 μg 3282.2 OSK1-IL-21 1000 μg  16484.1 OSK1-IL-22 500 μg 16050.1OSK1-IL-37 500 μg 3190.8 OSK1-IL-38 500 μg 13909.2 OSK1-IL-31 500 μg2067.7 OSK1-IL-32 500 μg 1559.4 OSK1-IL-33 500 μg 5859.0 OSK1-IL-39 500μg 6954.8 OSK1-IL-40 500 μg 5323.0 OSK1-IL-41 500 μg 8097.8

Example 9: Assessment of OSK-1-IL-17A Conjugates in Model Mice ofImiquimod-Induced Psoriasiform Dermatitis—Part 1

A model of psoriasiform dermatitis induced by imiquimod was used as apsoriasis model. Three types of OSK-1-IL-17A conjugates (OSK1-IL-31,OSK1-IL-32 and OSK1-IL-40) were intracutaneously administered to6-week-old BALB/c mice at a dose of 500 μg per animal 3 times at 2-weekintervals. At 2 weeks after the 3rd vaccination, the dorsal skin wasshaved and depilated with a depilatory cream. To the denuded skin,Beselna Cream 5% (trade name, Mochida Pharmaceutical Co., LTD.) wasapplied at a daily dose of 62.5 mg of imiquimod for 8 days for psoriasisinduction. To the ears, Beselna Cream 5% was applied at a daily dose of0.35 mg of imiquimod per ear for 8 days for psoriasis induction.

The skin conditions (erythema, the degree of induration, the area ofskin covered with silvery-white scale) were observed daily for 8 daysfrom the start of psoriasis induction (Day 0 to Day 7), and scored on a5-point scale of 0 to 4 (0: none, 1: mild, 2: moderate, 3: severe and 4:very severe) for assessment. The ear thickness was measured daily for 8days from the start of psoriasis induction (Day 0 to Day 7) using adigital caliper. The results of the skin conditions are shown in FIG.14, and the results of the ear thickness are shown in FIG. 15. Theresults show that the four types of OSK-1-IL-17A conjugates alleviateimiquimod-induced psoriasiform dermatitis.

Example 10: Assessment of OSK-1-IL-17A Conjugate in Model Mice ofImiquimod-induced Psoriasiform Dermatitis—Part 2 (1) Skin Condition andEar Thickness

OSK1-IL-21, which is an OSK-1-IL-17A conjugate, was intracutaneouslyadministered to Balb/c mice at a dose of 100 μg, 300 μg or 1000 μg peranimal 3 times at 2-week intervals. Separately, KLH-IL-21, which usedKLH as a carrier protein instead of OSK-1, was intracutaneouslyadministered to Balb/c mice at a dose of 20 μg per animal 3 times at2-week intervals. For the administration of KLH-IL-21, a mixture ofKLH-IL-21 with Freund's complete adjuvant (SIGMA) at 50 μL/mouse wasused at the first administration, and a mixture of KLH-IL-21 withFreund's incomplete adjuvant (SIGMA) at 50 μL/mouse was used at thesecond and third administrations. The procedures were the same as usedin Example 9 shown above except that the different vaccine was used andthat Beselna Cream 5% was applied daily for 13 days.

Photographs of skin conditions on Day 6 after the start of psoriasisinduction are shown in FIG. 16. Changes in the skin conditions duringthe period of psoriasiform dermatitis induction (Day 0 to Day 12) areshown in FIG. 17, and changes in the ear thickness during the period ofpsoriasiform dermatitis induction (Day 0 to Day 7) are shown in FIG. 18.The results show that the OSK-1-IL-17A conjugate is capable of morepotently alleviating imiquimod-induced psoriasiform dermatitis than theKLH-IL-17A conjugate.

(2) Immunohistochemical Staining

After the induction of psoriasiform dermatitis by application ofimiquimod, immune cells present in the skin and involucrin expression inthe skin were detected by immunohistochemical staining.

On Day 12 after the start of psoriasis induction, the dorsal skin andthe auricles were dissected, fixed with 4% PFA, and embedded inparaffin. After section preparation, deparaffinization and antigenretrieval treatment were performed. Then, the sections were blocked toprevent nonspecific reaction, reacted with a primary antibody, andreacted with a labeled secondary antibody. The sections were stained forinvolucrin, which is the final marker of keratinization, to examine thedegree of keratinization. The sections were also stained for themacrophage marker F4/80 and the granulocyte marker Gr-1 to detect immunecells.

The results of the involucrin staining are shown in FIG. 19, the resultsof the F4/80 staining are shown in FIG. 20, and the results of the Gr-1staining are shown in FIG. 21. As shown in these results, the expressionof involucrin, which was locally observed in the upper part of thestratum spinosum in the normal skin, was widely spread over theepidermis in the skin subjected to the application of imiquimod,representing a sign of enhanced keratinization. In the OSK-1-IL-17Aconjugate administration group, this phenomenon was remarkablysuppressed as compared with the KLH-IL-17A conjugate administrationgroup. In the skin and the auricles subjected to the application ofimiquimod, the number of macrophages, which are F4/80-positive cells,and the number of granulocytes, which are Gr-1-positive cells, wereincreased more remarkably. In the OSK-1-IL-17A conjugate administrationgroup, infiltration of these immune cells was remarkably inhibited ascompared with the KLH-IL-17A conjugate administration group.

(3) Blood IL-17 Concentration

Blood samples were collected before the start of psoriasis induction(Day 0) and on Day 12 after the start of psoriasis induction, and serumIL-17 concentration was measured using Mouse IL-17 A/F HeterodimerQuantikine ELISA Kit (R&D systems). The results are shown in FIG. 22.FIG. 22A shows the results of the mice before the application ofimiquimod (Day 0), and FIG. 22B shows the results of the mice subjectedto daily application of imiquimod (Day 12). As shown in FIG. 22B, in theOSK-1-IL-17A conjugate administration groups, the blood IL-17A/Fconcentrations were lower than that of the KLH-IL-17A conjugateadministration group. In addition, as shown in FIG. 22A, only in theKLH-IL-17A conjugate administration group, the blood IL-17A/Fconcentration was elevated before the application of imiquimod.

(4) Expression of Inflammation-Related Factors in Psoriasis Lesions

On Day 12 after the start of psoriasis induction, the skin lesions ofpsoriasis were dissected, and RNA was extracted from the skin tissueusing RNeasy Fibrous Tissue Mini Kit. The messenger RNA levels of theinflammation-related factors IL-17A, IL-17F, IL-22 and IL-23 weremeasured by real-time PCR using the primers and probe of TaqMan GeneExpression Assays.

As a result, the messenger RNA levels of the indicated factors werehigher in the mice not subjected to administration of the OSK-1-IL-17Aconjugate, but in the OSK-1-IL-17A conjugate administration groups, theexpression of the messenger RNAs of the indicated factors wassuppressed.

In Examples 9 and 10 shown above, model mice of imiquimod-inducedpsoriasiform dermatitis were used for the assessment of the medicalefficacy of the OSK-1-IL-17A conjugate, but model mice of IL-23-inducedpsoriasiform dermatitis were also used for the same purpose. Thespecific procedure is described in, for example, W. Jiang et al. (“AToll-Like Receptor 7, 8, and 9 Antagonist Inhibits Th1 and Th17Responses and Inflammasome Activation in a Model of IL-23-InducedPsoriasis” W. Jiang, et al., 1784 Journal of Investigative Dermatology(2013), Volume 133) and is as follows. Six to eight-week-old C57BL/6mice are prepared, and 0.5 mg of mouse IL-23 is dissolved in 20 mL ofPBS. An aliquot of the mouse IL-23 solution is intracutaneously injectedto the left ear of each mouse, for example, on Day 0, Day 2, Day 4, Day6, Day 10, Day 12 and Day 14 to induce dermatitis. The mice with induceddermatitis are randomly assigned to groups, and the test sample or acontrol sample (for example, PBS) is administered to the correspondinggroup of mice. The test sample or the control sample is subcutaneouslyadministered, for example, on Day 3, Day 6, Day 9, Day 12 and Day 15.The thickness of the auricle is measured with a digital caliper, forexample, on Day 18. In addition, the auricle is dissected andhistopathologically analyzed.

Example 11: Examination of Antibody Production Induced by OSK-1-IgEConjugate

An OSK-1-IgE conjugate composed of the OSK-1 peptide (SEQ ID NO: 1) anda mouse IgE epitope peptide (SEQ ID NO: 23) via an ε-Acp linker wasproduced and intracutaneously administered to Balb/c mice at a dose of100 or 250 μg per animal 3 times at 2-week intervals. Every 2 weeksafter the first administration, blood samples were collected, and theantibody titer against the epitope peptide was measured by ELISA.

The antibody titers measured 8 weeks after the first administration areshown in Table 5. The antibody titer is expressed as a half maximumvalue.

TABLE 5 IgE vaccine Half Maximum Half Maximum(8 W) ID 100 μg 250 μgOSK1-IgE-23 4093. 3 10487. 0

Example 12: Examination of Antibody Production Induced by OSK-1-PCSK9Conjugates

Three types of OSK-1-PCSK9 conjugates composed of the OSK-1 peptide (SEQID NO: 1) conjugated to a mouse PCSK9 epitope peptide (SEQ ID NO: 25, 26or 27) via an ε-Acp linker were produced and intracutaneouslyadministered to Balb/c mice at a dose of 100 or 250 μg per animal 3times at 2-week intervals. Every 2 weeks after the first administration,blood samples were collected, and the antibody titer against eachepitope peptide was measured by ELISA.

The antibody titers measured 8 weeks after the first administration areshown in Table 6. The antibody titer is expressed as a half maximumvalue.

TABLE 6 PCSK9 vaccine Half Maximum Half Maximum(8 W) ID 100 μg 250 μgOSK1-PCSK9-25 21.4  434.2 OSK1-PCSK9-26 2440.3 NT OSK1-PCSK9-27 1596.04632.1 NT: Not Tested

Example 13: Assessment of OSK-1-PCSK9 Conjugate by Blood PCSK9Concentration Measurement

The serum PCSK9 concentration was measured using the blood samplescollected from the mice to which OSK1-PCSK9-27 had been administered 3times at a dose of 250 μg (the OSK-1-PCSK9 conjugate administrationgroup) in Example 12. The blood samples were collected before the firstadministration and at 2, 4 and 6 weeks after the first administration.Separately, an OSK-1 administration group was prepared as a control, theadministration of OSK-1 and the collection of blood samples wereperformed on the same schedule as employed in the OSK-1-PCSK9 conjugateadministration group, and the serum PCSK9 concentration was measured.For the measurement of the serum PCSK9 concentration, Mouse ProproteinConvertase 9/PCSK9 Quantikine ELISA Kit (R&D systems) was used. It hasbeen previously reported that the administration of a PCSK9 antibodyinduces the elevation of blood PCSK9 concentration. This phenomenon isconsidered to result from the stabilization of the PCSK9 protein by theantibody, which slows the elimination of the PCSK9 protein from thebody. Based on this consideration, the elevation of the blood PCSK9level by vaccination can be regarded as an evidence for the fact thatthe antibody produced by vaccination acts on its target protein(Peptide-Based Anti-PCSK9 Vaccines—An Approach for Long-Term LDLcManagement, PLoS One. 2014; 9(12), An Anti-PCSK9 Antibody ReducesLDL-Cholesterol On Top Of A Statin And Suppresses HepatocyteSREBP-Regulated Genes; Int. J. Biol. Sci. 2012, 8(3): 310-327).

The results are shown in FIG. 23. In the OSK-1-PCSK9 conjugateadministration group, the blood PCSK9 levels at 2 to 6 weeks after thefirst administration were elevated from the level before the firstadministration.

Reference Example 3: Effect of KLH-PCSK9 Conjugate on Blood Lipid Levels

For this test, a KLH-PCSK9 conjugate composed of a mouse PCSK9 epitopepeptide (SEQ ID NO: 26) conjugated to KLH was used. Seven-week old ApoEdeficient mice were purchased from Charles River Japan. The mice wereassigned to a low-dose KLH-PCSK9 conjugate group (5 μg (PCSK9peptide)/mouse), a high-dose KLH-PCSK9 conjugate group (50 μg (PCSK9peptide)/mouse), a KLH administration group and a physiological salineadministration group as a control. The amount of KLH administered to themice of the KLH administration group was equal to the amount of KLHcontained in the conjugate administered to the mice of the conjugateadministration groups. The KLH-PCSK9 conjugate or KLH alone was mixedwith an equal volume of Freund's adjuvant (Wako Pure ChemicalIndustries, Ltd.) before administration and the mixture wassubcutaneously administered. Complete Freund's adjuvant was used at thefirst administration, and incomplete Freund's adjuvant was used at thesecond administration and later. The conjugates were administered 3times in total (Day 0, Day 14 and Day 28). At 24 weeks after the finaladministration, blood samples were collected, and the lipid levels weremeasured. As a result, in the KLH-PCSK9 conjugate administration groups,the levels of CM (chylomicron) and VLDL were reduced to nearly half, andthe level of TG (triglyceride) was reduced to nearly ⅔.

The present invention is not limited to the particular embodiments andexamples described above, and various modifications can be made withinthe scope of the appended claims. Other embodiments provided by suitablycombining technical means disclosed in separate embodiments of thepresent invention are also within the technical scope of the presentinvention. All the academic publications and patent literature cited inthe description are incorporated herein by reference.

1. A vaccine comprising a complex of a peptide consisting of an amino acid sequence that is the same or substantially the same as the amino acid sequence of SEQ ID NO: 1 and an epitope of a disease-causing biological protein.
 2. The vaccine according to claim 1, wherein the biological protein is one kind selected from the group consisting of DPP4, IL-17A, IgE, S100A9 and PCSK9.
 3. The vaccine according to claim 2, wherein the epitope of IL-17A is a peptide consisting of the amino acid sequence of any of SEQ ID NOs: 10, 11 and 29 to 36, or a peptide consisting of an amino acid sequence that is the same as the amino acid sequence of any of SEQ ID NOs: 10, 11 and 29 to 36 except for 1 or 2 amino acid deletions, substitutions or additions.
 4. The vaccine according to claim 2, wherein the epitope of DPP4 is a peptide consisting of the amino acid sequence of any of SEQ ID NOs: 2 to 9, or a peptide consisting of an amino acid sequence that is the same as the amino acid sequence of any of SEQ ID NOs: 2 to 9 except for 1 or 2 amino acid deletions, substitutions or additions.
 5. The vaccine according to claim 2, wherein the epitope of IgE is a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of an amino acid sequence that is the same as the amino acid sequence of SEQ ID NO: 12 except for 1 or 2 amino acid deletions, substitutions or additions.
 6. The vaccine according to claim 2, wherein the epitope of S100A9 is a peptide consisting of the amino acid sequence of SEQ ID NO: 13, or a peptide consisting of an amino acid sequence that is the same as the amino acid sequence of SEQ ID NO: 13 except for 1 or 2 amino acid deletions, substitutions or additions.
 7. The vaccine according to claim 2, wherein the epitope of PCSK9 is a peptide consisting of the amino acid sequence of any of SEQ ID NOs: 14 to 16, or a peptide consisting of an amino acid sequence that is the same as the amino acid sequence of any of SEQ ID NOs: 14 to 16 except for 1 or 2 amino acid deletions, substitutions or additions.
 8. The vaccine according to claim 2, wherein the epitope of the biological protein is conjugated to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 via ε-aminocaproic acid.
 9. The vaccine according to claim 2, wherein the amino acid at the N-terminus of the complex is acetylated.
 10. The vaccine according to claim 2, wherein the amino acid at the C-terminus of the complex is amidated.
 11. An immunogenic composition comprising a complex of a peptide consisting of an amino acid sequence that is the same or substantially the same as the amino acid sequence of SEQ ID NO: 1 and an epitope of a disease-causing biological protein.
 12. The immunogenic composition according to claim 11, wherein the biological protein is one kind selected from the group consisting of DPP4, IL-17A, IgE, S100A9 and PCSK9.
 13. A method for preventing or treating a disease caused by a biological protein, the method comprising administering, to an animal, an effective amount of a complex of a peptide consisting of an amino acid sequence that is the same or substantially the same as the amino acid sequence of SEQ ID NO: 1 and an epitope of the biological protein.
 14. A complex for use in prevention or treatment of a disease caused by a biological protein, the complex of a peptide consisting of an amino acid sequence that is the same or substantially the same as the amino acid sequence of SEQ ID NO: 1 and an epitope of the biological protein.
 15. (canceled)
 16. The vaccine according to claim 1, wherein the epitope of the biological protein is conjugated to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 via ε-aminocaproic acid.
 17. The vaccine according to claim 1, wherein the amino acid at the N-terminus of the complex is acetylated.
 18. The vaccine according claim 1, wherein the amino acid at the C-terminus of the complex is amidated. 